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Southern blot analysis I. (A) Southern analysis on XbaI/EcoRI digested gDNA derived from the parasite lines indicated. WR, WR99210; G, ganciclovir; ES, episomal plasmid; I, possible integration signal; E, endogenous gene locus. (B) Quantitative Southern blot to analyse copy number of 5 9 homology region. dhps, dihydropteroate synthase. P1A, P1B refer to signals resulting from hybridisation of probe 1; D, dhps. The ratio (P1A + P1B)/D for all clones was normalised against the ratio for wild-type 3D7 ( = 1). doi:10.1371/journal.pone.0018396.g003
Source publication
Upon invading the host erythrocyte, the human malaria parasite P. falciparum lives and replicates within a membrane bound compartment referred to as the parasitophorous vacuole. Recently, interest in this compartment and its protein content has grown, due to the important roles these play in parasite egress and protein traffic to the host cell. Sur...
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Context 1
... We analysed this parasite population by Southern blot, using a probe diagnostic for the specific integration event. A successful double crossover integration event would result in the disappearance of the endogenous 4.1 kbp fragment, and the detection of a 5.4 kbp band. As a control, we analysed in parallel the wild-type 3D7 parental strain (Fig. 3A, lane 1), and the parasite population containing the episomally maintained knockout vector (prior to ganciclovir selection, Fig. 3A, lane 2). This experiment revealed that, despite negative selection against the presence of the plasmid backbone (and thus TK expression cassette), clonal parasites still maintain the knockout plasmid as ...
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... double crossover integration event would result in the disappearance of the endogenous 4.1 kbp fragment, and the detection of a 5.4 kbp band. As a control, we analysed in parallel the wild-type 3D7 parental strain (Fig. 3A, lane 1), and the parasite population containing the episomally maintained knockout vector (prior to ganciclovir selection, Fig. 3A, lane 2). This experiment revealed that, despite negative selection against the presence of the plasmid backbone (and thus TK expression cassette), clonal parasites still maintain the knockout plasmid as an episome (Fig. 3A, lane 3). We were not able to detect any fragment migrating at 5.4 kbp, indicating that the pv1 locus was still ...
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... lane 1), and the parasite population containing the episomally maintained knockout vector (prior to ganciclovir selection, Fig. 3A, lane 2). This experiment revealed that, despite negative selection against the presence of the plasmid backbone (and thus TK expression cassette), clonal parasites still maintain the knockout plasmid as an episome (Fig. 3A, lane 3). We were not able to detect any fragment migrating at 5.4 kbp, indicating that the pv1 locus was still intact in these ...
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... Following drug cycling, we then added ganciclovir to select for double-crossover integration events. We monitored possible integration events using the diagnostic Southern blot detailed in the previous section. Encouragingly, parasite populations from the second and third drug cycle lost the episomal plasmid bands following ganciclovir treatment (Fig. 3A). Additionally, a fragment indicative of the correct integration event could also be detected (Fig. ...
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... monitored possible integration events using the diagnostic Southern blot detailed in the previous section. Encouragingly, parasite populations from the second and third drug cycle lost the episomal plasmid bands following ganciclovir treatment (Fig. 3A). Additionally, a fragment indicative of the correct integration event could also be detected (Fig. ...
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... parasite population resulting from 3 cycles of WR99210 selection followed by ganciclovir pressure, and analysed three independent clones, referred to as C2, C3 and E10. Diagnostic Southern blot revealed that all three clones, although apparently retaining the 4.1 kbp endogenous band, also showed a second fragment, migrating at between 5 and 6 kbp (Fig. 3A). Interestingly, whilst clones C3 and E10 showed a band at close to 6 kbp, clone C2 has obtained a fragment running at just over 5 kbp, close to the calculated size for an integration event. Nevertheless, retention of the endogenous 4.1 kbp fragment suggested that clones C2, C3 and E10 did not in fact represent a purely clonal ...
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... copy dhps gene as a control. We measured signal intensity derived from Probe1 and dhps, and set the the ratio derived from wild type 3D7 to 1 as a normalisation step. In all three clones studied, the normalised ratio (total signal Probe 1)/ (total signal Probe dhps) deviated considerably from 1, ranging from 1.8 (clone C2) to 2.5 (clone E10, Fig. 3B). Thus, clones C2, C3 and E10 contain a duplication event of at least the 59 homology region contained on the pHTKDPV1 ...
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Citations
... Treatment of erythrocytes infected with trophozoite-stage parasites with PBS, which leaves the erythrocyte intact, or SLO, which perforates the erythrocyte membrane and thereby releases exported proteins into the supernatant while leaving the PVM intact, did not release PV6, even though the soluble exported protein HRPII was released (Fig. 2C). In contrast, treatment of the parasite with saponin, which lyses the erythro cyte membrane and the PVM, released PV6, along with the previously characterized PV protein PV1 (51,52). This further indicates that PV6 is a PV-resident protein. ...
Malaria parasites alter multiple properties of the host erythrocyte by exporting proteins into the host cell. Many exported proteins contain a five-amino acid motif called the Plasmodium export element (PEXEL) that is cleaved by the parasite protease Plasmepsin V (PM V). The presence of a PEXEL is considered a signature of protein export and has been used to identify a large number of exported proteins. The export of proteins becomes essential midway through the intraerythrocytic cycle—preventing protein export blocks parasite development 18–24 h after invasion. However, a genetic investigation revealed that the absence of the PEXEL protein PFA0210c (PF3D7_0104200) causes parasite development to arrest immediately after invasion. We now show that this protein is cleaved by PM V but not exported into the host erythrocyte and instead functions in the parasitophorous vacuole; hence, the protein was renamed PV6. We additionally show that the lysine residue that becomes the N-terminus of PV6 after processing by PM V prevents export. This is the first example of a native Plasmodium falciparum PM V substrate that remains in the parasitophorous vacuole. We also provide evidence suggesting that the parasite may produce at least one additional essential, non-exported PM V substrate. Therefore, the presence of a PEXEL and, hence, processing of a protein by PM V do not always target a protein for export, and PM V likely has a broader function in parasite growth beyond processing exported proteins. Furthermore, we utilized this finding to investigate possible requirements for protein export further.
IMPORTANCE
In the manuscript, the authors investigate the role of the protease Plasmepsin V in the parasite–host interaction. Whereas processing by Plasmepsin V was previously thought to target a protein for export into the host cell, the authors now show that there are proteins cleaved by this protease that are not exported but instead function at the host–parasite interface. This changes the view of this protease, which turns out to have a much broader role than anticipated. The result shows that the protease may have a function much more similar to that of related organisms. The authors also investigate the requirements for protein export by analyzing exported and non-exported proteins and find commonalities between the proteins of each set that further our understanding of the requirements for protein export.
... The few known P. falciparum DG proteins enable transport of parasite effector proteins into the erythrocyte [27][28][29] or are exported and subsequently mediate alterations of the biochemical and biophysical properties of the host cell [30]. The only known contents of Plasmodium DGs are the five core proteins of the Plasmodium translocon of exported proteins (PTEX) -EXP2, HSP101, PTEX150, PTEX88 and TRX2 -an essential protein complex that transports parasite effector proteins across the PVM into the host cell [28,[31][32][33][34]; PV1, which interacts with exported proteins and the PTEX [35,36]; EXP1, a transmembrane protein in the parasitophorous vacuole membrane [37][38][39] that has an important role in nutrient uptake across the PVM through its effect on the localization of EXP2 [27,29,37,40]; P113, a glycosylphosphatidylinositol (GPI)-linked protein that associates with the PTEX and with PVM and exported proteins [41,42]; LSA3, the function of which is unknown [43]; and RESA (ring-infected erythrocyte surface antigen) [44], which binds to and stabilizes spectrin tetramers below the erythrocyte surface, thereby reducing host cell deformability [22,30,[45][46][47] (Table 1). The recently published Dense Granule Protein Database (DGPD) (http://dgpd.tlds.cc/DGPD/index/), ...
Malaria is an important infectious disease that continues to claim hundreds of thousands of lives annually. The disease is caused by infection of host erythrocytes by apicomplexan parasites of the genus Plasmodium. The parasite contains three different apical organelles - micronemes, rhoptries and dense granules (DGs) - whose contents are secreted to mediate binding to and invasion of the host cell and the extensive remodelling of the host cell that occurs following invasion. Whereas the roles of micronemes and rhoptries in binding and invasion of the host erythrocyte have been studied in detail, the roles of DGs in Plasmodium parasites are poorly understood. They have been proposed to control host cell remodelling through regulated protein secretion after invasion, but many basic aspects of the biology of DGs remain unknown. Here we describe DG biogenesis timing for the first time, using RESA localization as a proxy for the timing of DG formation. We show that DG formation commences approximately 37 min prior to schizont egress, as measured by the recruitment of the DG marker RESA. Furthermore, using a bioinformatics approach, we aimed to predict additional cargo of the DGs and identified the J-dot protein HSP40 as a DG protein, further supporting the very early role of these organelles in the interaction of the parasite with the host cell.
... Three mutants were found to dropout at different points of this experiment due to either reduced ABS growth rate or very low abundance in the pool following transfection: PBANKA_1425900 (P2), HADO (P7) and PV1 (P8). HADO encodes a putative magnesium phosphatase important for ookinete development, possibly by regulating the actin dynamics (Akinosoglou et al., 2015), and PV1 encodes a homolog of an essential protein of the P. falciparum parasitophorous vacuole (Chu et al., 2011), likely an accessory of the translocon protein complex PTEX (Morita et al., 2018). Another mutant that started with a relatively high abundance (0.048) but was significantly depleted from the parasite population from P5 onward was GAMER ( Figure 1D). ...
Malaria remains one of the most devastating infectious diseases. Reverse genetic screens offer a powerful approach to identify genes and molecular processes governing malaria parasite biology. However, sexual reproduction and complex regulation of gene expression and genotype-phenotype associations in the mosquito have hampered the development of screens in this key part of the parasite lifecycle. We designed a genetic approach in the rodent parasite Plasmodium berghei , which in conjunction with barcode sequencing allowed us to overcome the fertilization roadblock and screen for gametocyte-expressed genes required for parasite infection of the mosquito Anopheles coluzzii . The results confirmed previous findings, validating our approach for scaling up, and identified new genes required for ookinete motility and mosquito midgut infection and for sporozoite development and oocyst egress and salivary gland infection. Our findings can assist efforts to study malaria transmission biology and develop new interventions to control disease transmission.
... Plasmodium falciparum (3D7 strain) cells at the asexual stage were cultured in vitro in human erythrocytes (blood group O+) obtained from the Beijing Red Cross Blood Center. The parasite was grown under 5% O 2 and 5% CO 2 in RPMI-1640 medium supplemented with 5 g/l Albumax II (Life Technologies, Carlsbad, CA, USA), 2 g/l sodium bicarbonate, 25 mM HEPES (pH 7.4, adjusted with KOH), 1 mM hypoxanthine and 50 mg/L gentamicin, as previously described [18]. ...
Background
Haem is a key metabolic factor in the life cycle of the malaria parasite. In the blood stage, the parasite acquires host haemoglobin to generate amino acids for protein synthesis and the by-product haem for metabolic use. The malaria parasite can also synthesize haem de novo on its own. Plasmodium falciparum-specific histidine-rich protein 2 (PfHRP2) has a haem-binding site to mediate the formation of haemozoin, a biocrystallized form of haem aggregates. Notably, the gene regulates the mechanism of haemoglobin-derived haem metabolism and the de novo haem biosynthetic pathway in the Pfhrp2-disrupted parasite line during the intraerythrocytic stages.
Methods
The CRISPR/Cas9 system was used to disrupt the gene locus of Pfhrp2. DNA was extracted from the transgenic parasite, and PCR, Southern blotting and Western blotting were used to confirm the establishment of transgenic parasites. RNA-sequencing and comparative transcriptome analysis were performed to identify differences in gene expression between 3D7 and Pfhrp2⁻-3D7 parasites.
Results
Pfhrp2⁻ transgenic parasites were successfully established by the CRISPR/Cas9 system. A total of 964, 1261, 3138, 1064, 2512 and 1778 differentially expressed genes (DEGs) were identified in the six comparison groups, respectively, with 373, 520, 1499, 353, 1253 and 742 of these DEGs upregulated and 591, 741, 1639, 711, 1259 and 1036 of them downregulated, respectively. Five DEGs related to haem metabolism and synthesis were identified in the comparison groups at six time points (0, 8, 16, 24, 32, and 40 h after merozoite invasion). The genes encoding delta-aminolevulinic acid synthetase and ferrochelatase, both related to haem biosynthesis, were found to be significantly upregulated in the comparison groups, and those encoding haem oxygenase, stromal-processing peptidase and porphobilinogen deaminase were found to be significantly downregulated. No GO terms were significantly enriched in haem-related processes (Q value = 1).
Conclusion
Our data revealed changes in the transcriptome expression profile of the Pfhrp2⁻-3D7 parasite during the intraerythrocytic stages. The findings provide insight at the gene transcript level that will facilitate further research on and development of anti-malaria drugs.
... P. falciparum asexual stages (3D7 strain) were cultured in vitro in human erythrocytes (blood group O+) obtained from the Beijing Red Cross Blood Center. It was grown under 5% O 2 , and 5% CO 2 in RPMI-1640 media supplemented with 5 g/L Albumax II (Life Technologies), 2 g/L sodium bicarbonate, 25 mM HEPES pH7.4 (pH adjusted with potassium hydroxide), 1 mM hypoxanthine and 50 mg/L gentamicin as previously described [18] . ...
Background. Heme is a key metabolic factor in the life of malaria parasite. In the blood stage it acquires host hemoglobin to generate amino acids for its own protein synthesis and by-product heme for metabolic use. The malaria parasite also can de novo synthesize heme by itself. Plasmodium falciparum-specific histidine-rich protein 2 (PfHRP2) is a histidine-rich protein and has a heme-binding site to mediate hemozoin formation, a bio-crystallized form of heme-aggregates. It is interesting to investigate the vibration of hemoglobin-derived heme metabolism and de novo heme-biosynthetic pathway in the Pfhrp2 disrupted parasites during the intraerythrocytic stages.
Methods. A CRISPR-Cas9 system was used to disrupt the gene locus of Pfhrp2. DNA was extracted from the transgenic parasites and polymerase chain reaction (PCR), western blotting and southern blotting were used to manifest the successful establishment of transgenic parasites. RNA-seq and comparative transcriptome analysis were performed to identify the difference in gene expression between 3D7 and Pfhrp2⁻ 3D7 parasites.
Results. Pfhrp2⁻ transgenic parasites were successfully established by the CRISPR/Cas9 system. The disruption of the exon 2 of Pfhrp2 can down-regulate the gene expression of Pfhrp3 which involved in hemoglobin-derived heme metabolism. It also up-regulates the gene expression level of enzymes of heme biosynthesis.
Conclusion: These data support that although Pfhrp2 is a dispensable gene for intraerythrocytic stages parasite but heme metabolism’s stabilizing is very important. The disruption of Pfhrp2 can both affect the pathway of heme metabolism and biosynthesis. A co-operation mechanism may exist between the heme biosynthesis and metabolism pathways for parasite growth in blood stage.
... P. falciparum (3D7 strain) cells at the asexual stage were cultured in vitro in human erythrocytes (blood group O+) obtained from the Beijing Red Cross Blood Center. The parasite was grown under 5% O 2 and 5% CO 2 in RPMI-1640 medium supplemented with 5 g/L Albumax II (Life Technologies), 2 g/L sodium bicarbonate, 25 mM HEPES (pH 7.4, adjusted with potassium hydroxide), 1 mM hypoxanthine, and 50 mg/L gentamicin as previously described [18] . ...
Background. Haem is a key metabolic factor in the life cycle of the malaria parasite. In the blood stage, the parasite acquires host haemoglobin to generate amino acids for protein synthesis and the by-product haem for metabolic use. The malaria parasite can also synthesize haem de novo by itself. Plasmodium falciparum-specific histidine-rich protein 2 (PfHRP2) has a haem-binding site to mediate the formation of haemozoin, a biocrystallized form of haem aggregates. Notably, the gene regulates the mechanism of haemoglobin-derived haem metabolism and the de novo haem biosynthetic pathway in the Pfhrp2-disrupted parasite line during the intraerythrocytic stages.
Methods. The CRISPR/Cas9 system was used to disrupt the gene locus of Pfhrp2. DNA was extracted from the transgenic parasite, and polymerase chain reaction (PCR), Southern blotting and Western blotting were used to confirm the establishment of transgenic parasites. RNA-Seq and comparative transcriptome analysis were performed to identify differences in gene expression between 3D7 and Pfhrp2⁻ 3D7 parasites.
Results. Pfhrp2⁻ transgenic parasites were successfully established by the CRISPR/Cas9 system. A total of 964, 1261, 3138, 1064, 2512, and 1778 differentially expressed genes (DEGs) were identified in the six comparison groups, and a total of 373, 520, 1499, 353, 1253, and 742 of the DEGs were upregulated, and 591, 741, 1639, 711, 1259, and 1036 of the DEGs were downregulated, respectively. Five DEGs related to haem metabolism and synthesis were identified in the comparison groups of six time points (0, 8, 16, 24, 32, and 40 h after merozoite invasion). The genes encoding ALAS and FC, related to haem biosynthesis, were found to be significantly upregulated in the comparison groups, and the HO, SPP, and PBGD genes were found to be significantly downregulated. No GO terms were significantly enriched in haem-related processes (Q value=1).
Conclusion: In this study, our findings revealed changes in the transcriptome expression profile of the Pfhrp2⁻3D7 parasite during the intraerythrocytic stages. The results suggested that disruption of Pfhrp2 alters the parasite’s haem metabolic and biosynthesis pathways at the gene transcript level. A cooperative mechanism exists between the haem biosynthesis and metabolic pathways for parasite growth and survival in the blood stage. It is difficult to treat malaria patients by inhibiting only one pathway with traditional antimalarial drugs. The above findings provide insight at the gene transcript level for further research and development of anti-malaria drugs.
... Parasite culture, synchronization, and pellet collection P. falciparum (3D7 strain) cells at the asexual stage were cultured in vitro in human erythrocytes (blood group O+) obtained from the Beijing Red Cross Blood Center. The parasite was grown under 5% O 2 and 5% CO 2 in RPMI-1640 medium supplemented with 5 g/L Albumax II (Life Technologies), 2 g/L sodium bicarbonate, 25 mM HEPES (pH 7.4, adjusted with potassium hydroxide), 1 mM hypoxanthine, and 50 mg/L gentamicin as previously described [18] . ...
Background: Haem is a key metabolic factor in the life cycle of the malaria parasite. In the blood stage, the parasite acquires host haemoglobin to generate amino acids for protein synthesis and the by-product haem for metabolic use. The malaria parasite can also synthesize haem de novo by itself. Plasmodium falciparum-specific histidine-rich protein 2 (PfHRP2) has a haem-binding site to mediate the formation of haemozoin, a biocrystallized form of haem aggregates. Notably, the gene regulates the mechanism of haemoglobin-derived haem metabolism and the de novo haem biosynthetic pathway in the Pfhrp2-disrupted parasite line during the intraerythrocytic stages.
Methods: The CRISPR/Cas9 system was used to disrupt the gene locus of Pfhrp2. DNA was extracted from the transgenic parasite, and polymerase chain reaction (PCR), Southern blotting and Western blotting were used to confirm the establishment of transgenic parasites. RNA-Seq and comparative transcriptome analysis were performed to identify differences in gene expression between 3D7 and Pfhrp2⁻ 3D7 parasites.
Results: Pfhrp2⁻ transgenic parasites were successfully established by the CRISPR/Cas9 system. A total of 964, 1261, 3138, 1064, 2512, and 1778 differentially expressed genes (DEGs) were identified in the six comparison groups, and a total of 373, 520, 1499, 353, 1253, and 742 of the DEGs were upregulated, and 591, 741, 1639, 711, 1259, and 1036 of the DEGs were downregulated, respectively. Five DEGs related to haem metabolism and synthesis were identified in the comparison groups of six time points (0, 8, 16, 24, 32, and 40 h after merozoite invasion). The genes encoding ALAS and FC, related to haem biosynthesis, were found to be significantly upregulated in the comparison groups, and the HO, SPP, and PBGD genes were found to be significantly downregulated. No GO terms were significantly enriched in haem-related processes (Q value=1).
Conclusion: Our data revealed changes in the transcriptome expression profile of the Pfhrp2⁻3D7 parasite during the intraerythrocytic stages. The above findings provide insight at the gene transcript level for further research and development of anti-malaria drugs.
... Single clones consistently yielded bands during integration PCR indicating the presence of the modified GEXP18 locus, wild-type GEXP18 locus as well as episomal plasmid in a single P. falciparum genome (See: 2.2.2). Similar results have previously been obtained by others for other genes (such as PV1) and were interpreted as evidence for gene duplication (Chu et al. 2011;Maier et al. 2008). While this is an interesting phenomenon it also means that this cell line could not be used in further experiments, because the non-modified wild-type locus would compensate any effects elicited by the downregulation of the modified gene. ...
Propagation of Plasmodium parasites within erythrocytes is responsible for the majority of the symptoms associated with the human malaria disease. Survival of these parasites relies on remodelling of their host cells via exported proteins. These proteins are trafficked to various locations within the host cell in order to fulfil their function. In particular, the causative agent of the most virulent form of the human malaria disease –P. falciparum– is known to introduce drastic changes of their infected host cells. Investigation of parasite genes implicated in host cell remodelling and protein trafficking is critical for our understanding of the disease and might open up novel ways to combat malaria. However, analysis of essential genes involved in these processes requires the use of conditional knockout or knockdown system. Despite this there was a prolonged lack of genetic systems suitable to interrogate essential P. falciparum genes until recently, when several new systems were introduced. This includes the promising new glmS system which enables knockdown of a modified gene through the self-cleaving action of the glmS ribozyme triggered by addition of glucosamine. Here we apply glmS for conditional downregulation of four P. falciparum genes: PFA0660w, GEXP18, CBP1 and PfJ23. PFA0660w was modified using conventional single-crossover recombination, while modification of the other three genes was executed via selection-linked-integration, a new system for integration into the P. falciparum genome. PFA0660w, CBP1 and PfJ23 were successfully modified with a glmS-sequence and enabled reliable glucosamine-dependent downregulation. Modification of GEXP18 coincided with retention of the wild-type GEXP18 locus, which meant that these cell lines could not be used in further experiments. The cell lines generated by selection-linked-integration also displayed signs indicative of malfunction of the SKIP peptide. Addition of glucosamine to the glmS-modified PFA0660w, CBP1 and PfJ23 cell lines enabled downregulation of protein abundance to ~10% of the non-treated control. This had no effects on parasite morphology and viability. Furthermore, no differences in knob-morphology upon downregulation of PFA0660w or CBP1 were observed. Also downregulation of PFA0660w did not induce changes in parasite cytoadhesion. However, inactivation of PFA0660w via severe truncation caused drastically deformed, elongated knob morphologies. This demonstrates that the downregulation of PFA0660w achieved via glmS was not strong enough to cause the observed mutant phenotype upon truncation. Overall effectivity of the glmS system seemed to be highly gene-specific and should therefore be restricted to high-priority targets. Also alternative systems should be considered for the further dissection of essential P. falciparum genes. Truncation of PFA0660w induced fascinating changes in infected red blood cell morphology which could be further dissected using high-resolution microscopy methods.
... a previously reported bona fide PV protein is essential for the parasite growth 11 . A recent report indicates that PfPV1 co-precipitates with PTEX complex 12,13 , suggesting that PfPV1 constitutes PTEX accessory molecules 13 . ...
... The protein has neither PEXEL motif nor predicted functional domain. PfPV1 orthologs were identified in various plasmodium species including, P. vivax, P. knowlesi, P. chaubaudi and P. berghei 11 . Previous studies suggested that PfPV1 is essential for blood-stage parasite growth and localizes to PV 11,26 . ...
... PfPV1 orthologs were identified in various plasmodium species including, P. vivax, P. knowlesi, P. chaubaudi and P. berghei 11 . Previous studies suggested that PfPV1 is essential for blood-stage parasite growth and localizes to PV 11,26 . In order to characterize the protein further, we utilized wheat germ cellfree system (WGCFS; CellFree Sciences, Matsuyama, Japan) to synthesize recombinant PfPV1. ...
Upon invasion, Plasmodium falciparum exports hundreds of proteins across its surrounding parasitophorous vacuole membrane (PVM) to remodel the infected erythrocyte. Although this phenomenon is crucial for the parasite growth and virulence, elucidation of precise steps in the export pathway is still required. A translocon protein complex, PTEX, is the only known pathway that mediates passage of exported proteins across the PVM. P. falciparum Parasitophorous Vacuolar protein 1 (PfPV1), a previously reported parasitophorous vacuole (PV) protein, is considered essential for parasite growth. In this study, we characterized PfPV1 as a novel merozoite dense granule protein. Structured illumination microscopy (SIM) analyses demonstrated that PfPV1 partially co-localized with EXP2, suggesting the protein could be a PTEX accessory molecule. Furthermore, PfPV1 and exported protein PTP5 co-immunoprecipitated with anti-PfPV1 antibody. Surface plasmon resonance (SPR) confirmed the proteins' direct interaction. Additionally, we identified a PfPV1 High-affinity Region (PHR) at the C-terminal side of PTP5 where PfPV1 dominantly bound. SIM analysis demonstrated an export arrest of PTP5ΔPHR, a PTP5 mutant lacking PHR, suggesting PHR is essential for PTP5 export to the infected erythrocyte cytosol. The overall results suggest that PfPV1, a novel dense granule protein, plays an important role in protein export at PV.
... S1, C to E). We visualized the effect of 49c on PVM breakdown using parasites expressing a green fluorescent protein (GFP) fusion of the soluble PV protein PfPVI (PfPVI-GFP) (14). When the PVM ruptures, pores form in the iRBC membrane, leading to the disappearance of the GFP signal (15). ...
Plasmodium parasite entrance and exit
Sweats and fever are the hallmarks of malaria. Red blood cells are the replication factories for malaria parasites. Fever occurs when the parasites' merozoite stages burst en masse from red blood cells into the circulation. Nasamu et al. and Pino et al. discovered that two parasite proteases, plasmepsin IX and X, are essential for mass cell exit (see the Perspective by Boddey). Plasmepsin X is also used by the merozoites to enter a fresh red blood cell to continue the replicative cycle. These two plasmepsins act by regulating the maturation of enzymes required to disrupt host cell membranes. Because these functions are essential for the parasite, the authors used protease inhibitors to show that plasmepsins provide potential drug targets.
Science , this issue p. 518 , p. 522 ; see also p. 445
